Project Summary Acute respiratory distress syndrome (ARDS) is a clinical syndrome characterized by rapid-onset bilateral pulmonary infiltrates and acute respiratory failure hallmarked by an inflammatory response within the alveolar space. Damage to the alveolar epithelium causes disruption of the epithelial barrier, which in turn affects efficient gas exchange, fluid clearance, and host immune responses. Recovery from injury is an active process and occurs by recruitment of anti-inflammatory cells and mediators. Foxp3+ regulatory T cells (Tregs) contribute to the recovery from experimental acute lung injury (ALI) through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. Moreover, Tregs are present in the bronchoalveolar lavage fluid of patients with ARDS. There are still many unanswered questions regarding the mechanisms by which Tregs act to promote lung resolution. Our data show that transcriptional profile of Tregs isolated from lungs during the resolving phase of LPS-induced lung injury contains many differentially expressed genes, when compared to Tregs isolated from uninjured control lungs. These differences likely underlie both a immune suppressive response by Tregs and their tissue reparative functions, highlighting the complexity of Treg function in resolution and repair of lung injury. One transcript which was markedly upregulated in Tregs from resolving lungs is matrix metalloproteinase 12 (Mmp12). The role of Treg-expressed MMP12 in regulating turnover of the extracellular matrix, in processing chemotactic signals that promote ALI resolution, in other described MMP functions, and in novel functions not yet reported is not known. The localization of Tregs within the alveoli may result in specific and important functions for Treg MMP12 within these microenvironments. The hypothesis of this proposal is that expression of MMP12 by Tregs plays an important role in the resolution of acute lung injury. Aim 1 investigates the function of Treg-expressed MMP12 during ALI resolution. We will test the hypothesis that Treg expression of MMP12 is an important component of the Treg armamentarium in ALI resolution and will determine its functions. These studies use a transgenic mouse strain generated by breeding mice with a floxed Mmp12 exon to the Foxp3Cre-YFP strain, producing mice in which MMP12 is knocked out only in Tregs. Treg-expressed MMP12 will be studied during resolution of two lung injuries, LPS and influenza A H1N1 strain PR8. Aim 2 determines which inflammatory cues elicit Treg MMP12 expression. Our hypothesis is that the initial cytokine and chemokine responses in lung injury influence Treg expression of MMP12. We will examine this hypothesis in vitro with primary mouse Tregs. The results will enhance understanding of the reparative process in resolution of injury and may uncover potential new targets that both accelerate the recovery from ARDS and facilitate better quality lung repair. Funding from this R03 award will contribute to my path toward independence by allowing me to pursue a new line of investigation that arose from my K08 award. The proposed studies will expand the depth and breadth of my research in the resolution of ARDS/ALI and facilitate my upcoming R01 application.